Josep Casadesús

Host adapted serotypes of Salmonella enterica.

Salmonella constitutes a genus of zoonotic bacteria of worldwide economic and health importance. The current view of salmonella taxonomy assigns the members of this genus to two species: S. enterica and S. bongori. S. enterica itself is divided into six subspecies, enterica, salamae, arizonae, diarizonae, indica, and houtenae, also known as subspecies I, II, IIIa, IIIb, IV, and VI, respectively [1]. Members of Salmonella enterica subspecies enterica are mainly associated with warm-blooded vertebrates and are usually transmitted by ingestion of food or water contaminated by infected faeces. The pathogenicity of most of the distinct serotypes remains undefined, and even within the most common serotypes, many questions remain to be answered regarding the interactions between the organism and the infected host.Salmonellosis manifests itself in three major forms: enteritis, septicaemia, and abortion, each of which may be present singly or in combination, depending on both the serotype and the host involved. Although currently over 2300 serovars of Salmonella are recognized, only about 50 serotypes are isolated in any significant numbers as human or animal pathogens [2, 3] and they all belong to subspecies enterica. Of these, most cause acute gastroenteritis characterized by a short incubation period and a severe systemic disease in man or animals, characterized by septicaemia, fever and/or abortion, and such serotypes are often associated with one or few host species [4–6].It is the intention of this review to present a summary of current knowledge of these host-adapted serotypes of S. enterica. The taxonomic relationships between the serotypes will be discussed together with a comparison of the pathology and pathogenesis of the disease that they cause in their natural host(s). Since much of our knowledge on salmonellosis is based on the results of work on Typhimurium, this serotype will often be used as the baseline in discussion. It is hoped that an appreciation of the differences that exist in the way these serotypes interact with the host will lead to a greater understanding of the complex host–parasite relationship that characterizes salmonella infections.

Epigenetic Gene Regulation in the Bacterial World

SUMMARY Like many eukaryotes, bacteria make widespread use of postreplicative DNA methylation for the epigenetic control of DNA-protein interactions. Unlike eukaryotes, however, bacteria use DNA adenine methylation (rather than DNA cytosine methylation) as an epigenetic signal. DNA adenine methylation plays roles in the virulence of diverse pathogens of humans and livestock animals, including pathogenic Escherichia coli, Salmonella, Vibrio, Yersinia, Haemophilus, and Brucella. In Alphaproteobacteria, methylation of adenine at GANTC sites by the CcrM methylase regulates the cell cycle and couples gene transcription to DNA replication. In Gammaproteobacteria, adenine methylation at GATC sites by the Dam methylase provides signals for DNA replication, chromosome segregation, mismatch repair, packaging of bacteriophage genomes, transposase activity, and regulation of gene expression. Transcriptional repression by Dam methylation appears to be more common than transcriptional activation. Certain promoters are active only during the hemimethylation interval that follows DNA replication; repression is restored when the newly synthesized DNA strand is methylated. In the E. coli genome, however, methylation of specific GATC sites can be blocked by cognate DNA binding proteins. Blockage of GATC methylation beyond cell division permits transmission of DNA methylation patterns to daughter cells and can give rise to distinct epigenetic states, each propagated by a positive feedback loop. Switching between alternative DNA methylation patterns can split clonal bacterial populations into epigenetic lineages in a manner reminiscent of eukaryotic cell differentiation. Inheritance of self-propagating DNA methylation patterns governs phase variation in the E. coli pap operon, the agn43 gene, and other loci encoding virulence-related cell surface functions.

N6-methyl-adenine: an epigenetic signal for DNA-protein interactions.

N6-methyl-adenine is found in the genomes of bacteria, archaea, protists and fungi. Most bacterial DNA adenine methyltransferases are part of restriction–modification systems. Certain groups of Proteobacteria also harbour solitary DNA adenine methyltransferases that provide signals for DNA–protein interactions. In γ-proteobacteria, Dam methylation regulates chromosome replication, nucleoid segregation, DNA repair, transposition of insertion elements and transcription of specific genes. In Salmonella, Haemophilus, Yersinia and Vibrio species and in pathogenic Escherichia coli, Dam methylation is required for virulence. In α-proteobacteria, CcrM methylation regulates the cell cycle in Caulobacter, Rhizobium and Agrobacterium, and has a role in Brucella abortus infection.

Loss of Hfq activates the σE-dependent envelope stress response in Salmonella enterica

Ubiquitous RNA‐binding protein Hfq mediates the regulatory activity of many small RNAs (sRNAs) in bacteria. To identify potential targets for Hfq‐mediated regulation in Salmonella, we searched for lacZ translational fusions whose activity varied in the presence or absence of Hfq. Fusions downregulated by Hfq were more common than fusions showing the opposite response. Surprisingly, in a subset of isolates from the major class, the higher activity in the absence of Hfq was due to transcriptional activation by the alternative sigma factor RpoE (σE). Activation of the σE regulon normally results from envelope stress conditions that elicit proteolytic cleavage of the anti‐σE factor RseA. Using an epitope tagged variant of RseA, we found that RseA is cleaved at an increased rate in a strain lacking Hfq. This cleavage was dependent on the DegS protease and could be completely prevented upon expressing the hfq gene from an inducible promoter. Thus, loss of Hfq function appears to affect envelope biogenesis in a way that mimics a stress condition and thereby induces the σE response constitutively. In a RseA mutant, activation of the σE response causes Hfq‐dependent downregulation of outer membrane protein (OMP) genes including lamB, ompA, ompC and ompF. For ompA, downregulation results in part from σE‐dependent accumulation of MicA (SraD), a small RNA recently shown to downregulate ompA transcript levels in stationary phase. We show that the micA gene is under σE control, and that DegS‐mediated σE release is required for the accumulation of MicA RNA upon entry into stationary phase. A similar mechanism involving additional, still unidentified, sRNAs, might underlie the growth phase‐dependent regulation of other OMP mRNAs.

Salmonella enterica Serovar Typhimurium Response Involved in Attenuation of Pathogen Intracellular Proliferation

ABSTRACT Salmonella enterica serovar Typhimurium proliferates within cultured epithelial and macrophage cells. Intracellular bacterial proliferation is, however, restricted within normal fibroblast cells. To characterize this phenomenon in detail, we investigated the possibility that the pathogen itself might contribute to attenuating the intracellular growth rate. S.enterica serovar Typhimurium mutants were selected in normal rat kidney fibroblasts displaying an increased intracellular proliferation rate. These mutants harbored loss-of-function mutations in the virulence-related regulatory genes phoQ,rpoS, slyA, and spvR. Lack of a functional PhoP-PhoQ system caused the most dramatic change in the intracellular growth rate. phoP- andphoQ-null mutants exhibited an intracellular growth rate 20- to 30-fold higher than that of the wild-type strain. This result showed that the PhoP-PhoQ system exerts a master regulatory function for preventing bacterial overgrowth within fibroblasts. In addition, an overgrowing clone was isolated harboring a mutation in a previously unknown serovar Typhimurium open reading frame, namedigaA for intracellular growth attenuator. Mutations in other serovar Typhimurium virulence genes, such as ompR,dam, crp, cya,mviA, spiR (ssrA), spiA, and rpoE, did not result in pathogen intracellular overgrowth. Nonetheless, lack of either SpiA or the alternate sigma factor RpoE led to a substantial decrease in intracellular bacterial viability. These results prove for the first time that specific serovar Typhimurium virulence regulators are involved in a response designed to attenuate the intracellular growth rate within a nonphagocytic host cell. This growth-attenuating response is accompanied by functions that ensure the viability of intracellular bacteria.

Role for Salmonella enterica enterobacterial common antigen in bile resistance and virulence.

Passage through the digestive tract exposes Salmonella enterica to high concentrations of bile salts, powerful detergents that disrupt biological membranes. Mutations in the wecD or wecA gene, both of which are involved in the synthesis of enterobacterial common antigen (ECA), render S. enterica serovar Typhimurium sensitive to the bile salt deoxycholate. Competitive infectivity analysis of wecD and wecA mutants in the mouse model indicates that ECA is an important virulence factor for oral infection. In contrast, lack of ECA causes only a slight decrease in Salmonella virulence during intraperitoneal infection. A tentative interpretation is that ECA may contribute to Salmonella virulence by protecting the pathogen from bile salts.

DNA Adenine Methylation Regulates Virulence Gene Expression in Salmonella enterica Serovar Typhimurium

Transcriptomic analyses during growth in Luria-Bertani medium were performed in strain SL1344 of Salmonella enterica serovar Typhimurium and in two isogenic derivatives lacking Dam methylase. More genes were repressed than were activated by Dam methylation (139 versus 37). Key genes that were differentially regulated by Dam methylation were verified independently. The largest classes of Dam-repressed genes included genes belonging to the SOS regulon, as previously described in Escherichia coli, and genes of the SOS-inducible Salmonella prophages ST64B, Gifsy-1, and Fels-2. Dam-dependent virulence-related genes were also identified. Invasion genes in pathogenicity island SPI-1 were activated by Dam methylation, while the fimbrial operon std was repressed by Dam methylation. Certain flagellar genes were repressed by Dam methylation, and Dam(-) mutants of S. enterica showed reduced motility. Altered expression patterns in the absence of Dam methylation were also found for the chemotaxis genes cheR (repressed by Dam) and STM3216 (activated by Dam) and for the Braun lipoprotein gene, lppB (activated by Dam). The requirement for DNA adenine methylation in the regulation of specific virulence genes suggests that certain defects of Salmonella Dam(-) mutants in the mouse model may be caused by altered patterns of gene expression.

Repair of DNA Damage Induced by Bile Salts in Salmonella enterica

Exposure of Salmonella enterica to sodium cholate, sodium deoxycholate, sodium chenodeoxycholate, sodium glychocholate, sodium taurocholate, or sodium glycochenodeoxycholate induces the SOS response, indicating that the DNA-damaging activity of bile resides in bile salts. Bile increases the frequency of GC → AT transitions and induces the expression of genes belonging to the OxyR and SoxRS regulons, suggesting that bile salts may cause oxidative DNA damage. S. enterica mutants lacking both exonuclease III (XthA) and endonuclease IV (Nfo) are bile sensitive, indicating that S. enterica requires base excision repair (BER) to overcome DNA damage caused by bile salts. Bile resistance also requires DinB polymerase, suggesting the need of SOS-associated translesion DNA synthesis. Certain recombination functions are also required for bile resistance, and a key factor is the RecBCD enzyme. The extreme bile sensitivity of RecB−, RecC−, and RecA− RecD− mutants provides evidence that bile-induced damage may impair DNA replication.

Conjugal transfer of the virulence plasmid of Salmonella enterica is regulated by the leucine‐responsive regulatory protein and DNA adenine methylation

Host‐encoded functions that regulate the transfer operon (tra) in the virulence plasmid of Salmonella enterica (pSLT) were identified with a genetic screen. Mutations that decreased tra operon expression mapped in the lrp gene, which encodes the leucine‐responsive regulatory protein (Lrp). Reduced tra operon expression in an Lrp− background is caused by lowered transcription of the traJ gene, which encodes a transcriptional activator of the tra operon. Gel retardation assays indicated that Lrp binds a DNA region upstream of the traJ promoter. Deletion of the Lrp binding site resulted in lowered and Lrp‐independent traJ transcription. Conjugal transfer of pSLT decreased 50‐fold in a Lrp− background. When a FinO− derivative of pSLT was used, conjugal transfer from an Lrp− donor decreased 1000‐fold. Mutations that derepressed tra operon expression mapped in dam, the gene encoding Dam methyltransferase. Expression of the tra operon and conjugal transfer remain repressed in an Lrp− Dam− background. These observations support the model that Lrp acts as a conjugation activator by promoting traJ transcription, whereas Dam methylation acts as a conjugation repressor by activating FinP RNA synthesis. This dual control of conjugal transfer may also operate in other F‐like plasmids such as F and R100.

Distribution of insertion sequence IS200 in Salmonella and Shigella

Two DNA probes for the detection of insertion sequence IS200 by either Southern blotting or colony hybridization were constructed. One of the probes is a 300 bp EcoRI-HindIII fragment of IS200 cloned onto pBluescript KS(+); the other is a tail-to-tail dimer of the same fragment cloned onto pUC19. A survey of the presence of IS200 among enteric bacteria revealed that more than 90% of the pathogenic or food-poisoning isolates of Salmonella spp. examined contained one or more copies of insertion sequence IS200, with the exception of the subgenus I serovar S. agona in which IS200 is not found. Although insertion sequence IS200 was first considered a Salmonella-specific element, it also exists in many isolates of Shigella sonnei and Shigella flexneri, but not in Shigella dysenteriae.